Enzymes are used to process a variety of agricultural products such as wood, fruits and vegetables, starches, juices, and the like. Typically, processing enzymes are produced and recovered on an industrial scale from various sources, such as microbial fermentation (Bacillus α-amylase), or isolation from plants (coffee β-galactosidase or papain from plant parts). Enzyme preparations are used in different processing applications by mixing the enzyme and the substrate under the appropriate conditions of moisture, temperature, time, and mechanical mixing such that the enzymatic reaction is achieved in a commercially viable manner. One area where enzymes play an important role is in the area of corn milling.
Today corn is milled to obtain cornstarch and other corn-milling co-products such as corn gluten feed, corn gluten meal, and corn oil. The starch obtained from the process is often further processed into other products such as derivatized starches and sugars, or fermented to make a variety of products including alcohols or lactic acid.
The process of starch recovery from corn grain is well known and involves a wet-milling process. Corn wet-milling involves many time consuming and costly steps, which include steeping the corn kernel, grinding the corn kernel, and separating the components of the kernel. Dry-mill processes that make fermentable sugars (and then ethanol, for example) from cornstarch facilitate efficient contacting of exogenous enzymes with starch. These processes are less capital intensive than wet-milling but significant cost advantages are still desirable, as often the co-products derived from these processes are not as valuable as those derived from wet-milling.
Thus, for dry milling, there is a need for a method that improves the efficiency of the process and/or increases the value of the co-products. For wet milling, there is a need for a method of processing starch that does not require the equipment necessary for prolonged steeping, grinding, milling, and/or separating the components of the kernel. For example, there is a need to modify or eliminate the steeping step in wet milling as this would reduce the amount of waste water requiring disposal, thereby saving energy and time, and increasing mill capacity (kernels would spend less time in steep tanks). There is also a need to eliminate or improve the process of separating the starch-containing endosperm from the embryo.
The present invention relates to a transgenic corn (Zea mays) plant, event 3272 (Johnson et al, U.S. Pat. No. 7,635,799; incorporated by reference), that has incorporated into its genome a synthetic α-amylase gene (amy797E), encoding a thermostable Amy797E α-amylase capable of processing starch in plants. α-amylase enzymes act on starch and related polysaccharides and oligosaccharides in a random manner, performing endohydrolysis of (1→4)-α-D-glucosidic linkages in polysaccharides containing three or more (1→4)-α-linked D-glucose units, such as those found in starch. Upon expression and activation of the Amy797E α-amylase, the enzyme processes the starch substrate found within the plant or plant tissue. This processing results in an altered composition which facilitates plant and grain processing for milling, thereby making a significant improvement in processing corn plants or plant parts for fermentation compared to corn plants which do not express amy797E (see, for example, Lanahan et al., U.S. Pat. No. 7,102,057 and Batie et al., U.S. Pat. No. 7,914,993, which are incorporated by reference).
A highly sensitive method for detecting the Amy797E α-amylase from transgenic events is necessary for use in environmental monitoring, monitoring traits in crops in the field, or monitoring products derived from a crop harvest, as well as for use in ensuring compliance of parties subject to contractual terms. It is ideal to have a limit of detection of Amy797E protein of less than 0.1%. In other words, a method of detection is needed to detect less than one kernel of corn event 3272 per 1000 kernels.
Additionally, certain applications of milling of event 3272 require mixing event 3272 corn or corn seed with corn or corn seed that is not event 3272 (see, for example, U.S. Pat. No. 7,914,993). This mixing is required so that an optimal amount of Amy797E α-amylase per unit of corn or corn seed is achieved. A highly sensitive method of Amy979E detection would also benefit users who need to measure the amount of Amy797E present in a given amount of event 3272 corn or corn seed.
It is standard in the art to detect the presence of a specific polypeptide of interest using an antibody specific to the polypeptide of interest in an ELISA (enzyme-linked immunosorbent assay). An ELISA is a method for detecting a polypeptide of interest in a biological sample, the method typically comprising: (a) extracting protein from a biological sample; (b) assaying the extracted protein using an immunological method comprising an antibody specific for the polypeptide of interest; and (c) detecting the binding of said antibody to the polypeptide of interest. Detection is typically measured via an enzyme which is linked to a secondary antibody. The enzyme can metabolize a colorless substrate into a colored product. The optical density is measured, and this is proportional to the amount of colored product and to the amount of the polypeptide of interest present in the sample.
The ELISA method described above is known to one skilled in the art for the detection of transgenic polypeptides in plants. However, the polypeptide of interest here, Amy797E, presents an unusual challenge because its native substrate, starch, is found within the plant cell, and Amy797E tends to be pre-bound to its substrate in an inactive form. Starch is typically insoluble and comes out in the insoluble fraction during standard purification. Using the insoluble fraction for ELISA is undesirable, because the insoluble fraction significantly reduces the availability of the polypeptides, enzymes or other substrates bound or otherwise associated thereto for ELISA. Although not all of Amy797E in the plant cell is pre-bound to insoluble starch, it is preferred to release this pre-bound Amy797E to increase the limit of quantification and the limit of detection in a standard ELISA. The present invention solves this problem.